DNA methylation is an essential regulator of transcription, chromatin structure, development, and genomic stability in mammalian cells that is mediated by a family of DNA methyltransferases (DNMTs): DNMT1, DNMT3A, and DNMT3B. While normal patterns are critical for mammalian growth and development, deregulated DNA methylation patterns, characterized by global hypomethylation and gene-specific hypermethylation, are a hallmark of tumor cells and an early event in tumorigenesis. Recent exciting new findings, including those from our laboratory, pinpoint important links between the DNA methylation machinery and the repressive polycomb group (PcG) histone modifying complexes. PcG complexes PRC1 and PRC2 mediate histone H2AK119 monoubiquitination and H3K27 trimethylation, respectively and these complexes are also essential for growth and, when deregulated, contribute to cancer. The central hypothesis to be tested in this application is that de novo methyltransferase DNMT3B is a major regulator of genomic DNA methylation patterns in normal cells and that disruption of its functions contributes to DNA methylation defects in cancer. We propose that DNMT3B and polycomb repressive complex PRC1 cooperate to establish and maintain chromatin structure and normal gene regulation, while defects in their interaction and/or the posttranslational modifications mediated by PRC1 result in the aberrant DNA methylation defects characteristic of tumor cells. The following three aims are designed to test this hypothesis. In aim 1 we will dissect interactions between DNMT3B and PcG complexes and will characterize the function of a novel DNMT3B-PRC1/CBX4 interaction we have identified (and the post-translational modifications it mediates) using a comprehensive panel of in vitro assays. In aim 2 we will test whether DNMT3B and PRC1/CBX4 exert reciprocal effects on each other in vivo both at specific gene promoters and globally. Finally, in aim 3 we will determine genome-wide binding of DNMT3B in normal colon and colorectal cancer and will correlate these data with patterns of DNA methylation and histone marks to define epigenetic signatures in normal cells that may, in turn, help predict targets of aberrant DNA hypermethylation in colon cancer. Addressing this hypothesis will contribute to our fundamental understanding of how genomic DNA methylation patterns, particularly those mediated by DNMT3B, are established, maintained, and interface with aspects of the histone code. This is expected to positively affect human health by allowing for the development of novel therapies that target aberrant epigenetic changes and provide new diagnostic and prognostic markers to detect cancer earlier and more effectively. PUBLIC HEALTH RELEVANCE: Epigenetic modifications of the human genome, such as the addition of methyl groups to DNA (i.e. DNA methylation), are critical regulators of development and gene expression that frequently become deregulated in diseases like cancer and directly contribute to the growth of tumor cells. We lack a complete understanding of how DNA methylation patterns are established in normal cells and how these marks become disrupted in disease because the cellular machinery regulating these epigenetic marks has not been sufficiently well characterized. The main goal of this application is to gain a better understanding of how the enzymes regulating DNA methylation carry out their functions in vitro and in vivo and interact with other classes of epigenetic modifications in normal and cancerous cells.